Interleukin-1 (IL-1) is a proinflammatory cytokine that has several effects in the inflammation process. Stimulation of cells with IL-1 initiates a cascade of signaling events, including activation of c-Jun N-terminal kinase (JNK) and of nuclear transcription factor κB (NF-κB), which up-regulates the expression of many proinflammatory genes in the nucleus (Dinarello, Blood 87:2095-2147, 1996). In unstimulated cells, NF-κB is sequestered in the cytoplasm in a complex with inhibitory proteins I κB. Following stimulation with cytokines and other extracellular stimuli, the I κB proteins are phosphorylated on specific serine residues, which trigger the ubiquitination and subsequent degradation of I κB through the proteasome pathway. Proteolysis of I κB release NF-κB to translocate into the nucleus, where it stimulates transcription of specific target genes (Thanos et al., Cell 80:529-532, 1995; Verma et al., Genes Dev. 9:2723-2735, 1995; Baeuerle et al. Cell 87:13-20, 1996).
Recent studies have provided a model for how the IL-1 signal transduction cascade is regulated. The first signaling event for IL-1 is a ligand-induced complex formation of the type I receptor (IL-1RI) and the receptor accessory protein (IL-1RAcP) (Greenfeder et al., J. Biol. Chem. 270:13757-13765, 1995; Huang et al., Proc. Natl. Acad. Sci. USA 94:12829-12832, 1997; Korherr et al., Eur. J. Immunol. 27:262-267, 1997; Wesche et al., J. Biol. Chem. 272:7727-7731, 1997). The cytosolic myeloid differentiation protein MyD88 is next recruited to this complex (Cao et al., Science 271:1128-1131, 1996; Muzio et al., Science 278:1612-1615, 1997; Wesche et al., Immunity 7:837-847, 1997; Burns et al., J. Biol. Chem. 273:12203-12209, 1998), which in turn enables the association of the serine/threonine IL-1 receptor-associated kinase (IRAK). IRAK becomes highly phosphorylated, leaves the receptor complex, and interacts with TRAF6 (TNF receptor-associated factor 6), which is required for IL-1-induced JNK and NF-κB activation (Cao et al., Nature 383:443-446, 1996; Yamin et al., J. Biol. Chem. 272:21540-21547, 1997; Lomaga et al., Genes Dev. 13:1015-1024, 1999). Another serine/threonine kinase, NF-κB-inducing kinase (NIK), is believed to be a downstream component in activating NF-κB, but not in the JNK activation, in response to IL-1 (Malinin et al., Nature 385:540-544, 1997). Recently, two IκB kinases (IKKα/IKK1 and IKKβ/IKK2) have been implicated in signal-induced phosphorylation of the IκB proteins (DiDonato et al., Nature 388:548-554, 1997; Mercurio et al., Science 278:860-866, 1997; Regnier et al., Cell 90:373-383, 1997; Woronicz et al., Science 278:866-869, 1997; Zandi et al., Cell 91:243-252, 1997). The IKKs are components of a large complex, which contain NEMO (NF-κB essential modulator)/IKKγ (Rothwarf et al., Nature 395:297-300, 1998; Yamaoka et al., Cell 93:1231-1240, 1998). The present inventors have recently demonstrated that the protein kinase TAK1 is involved in the IL-1 signaling pathway (Ninomiya-Tsuji et al., Nature 398:252-256, 1999). Following exposure of the cells to IL-1, endogenous TAK1 is recruited to the TRAF6 complex, where it becomes activated. Activated TAK1 then stimulates a MAP kinase cascade leading to JNK activation and a NIK-IKK cascade leading to NF-κB activation. Thus, TAK1 is positioned downstream of TRAF6 in the IL-1-activated signaling cascade. This suggests that the bifurcation of the IL-1-induced JNK and NF-κB activation pathways occurs at the level of TAK1.
TAK1 was originally identified as a MAP kinase kinase kinase (MAPKKK) that is activated by transforming growth factor-β (TGF-β) family ligands. The present inventors have previously demonstrated that TAK1 functions in TGF-β signaling pathways in mammalian cells (Yamaguchi et al., Science 270:2008-2011, 1995; Shibuya et al., Science 272:1179-1182, 1996). In early Xenopus embryos TAK1 also participates in mesoderm induction and patterning meditated by bone morphogenetic protein (BMP), a TGF-β family ligand (Shibuya et al., EMBO J. 17:1019-1028, 1998; Yamaguchi et al., EMBO J. 18:179-187, 1999). Furthermore, the inventors have recently found that TAK1 is involved in the MAP kinase-like pathway that negatively regulates the Wnt signaling pathway (Ishitani et al., Nature 399:798-802, 1999). Thus, the fact that TAK1 has the capacity to activate distinct pathways raises the problem of how specificity in signaling pathways is achieved. The identification of specific components in different signaling pathways provides insight into the mechanisms for selective activation of TAK1 in response to divergent stimuli.
There are several groups of MAPKKKs including the Raf family (Raf-1 and B-Raf), the MEKK family (MEKK1, MEKK2, and MEKK3), the MLK family (MLK1, MLK2, MLK3, and DLK), as well as MTK1/MEKK4 and ASK1 (Fanger et al., Curr. Opin. Genet. Dev. 7:67-74, 1997; Robinson et al., Curr. Opin. Cell Biol. 9:180-186, 1997). Different mechanisms of MAPKKKs activation have been reported. Autophosphorylation mediated by an intra-molecular reaction has been implicated in the activation of MEKK1 and SSK2, a MAPKKK in budding yeast (Deak et al., Biochem. J. 322:185-192, 1997; Siow et al., J. Biol. Chem. 272:7586-7594, 1997; Posas et al., EMBO J. 17:1385-1394 1998). ASK1 and MLK3 have been demonstrated to form dimers in response to upstream stimuli, and this dimerization has been shown to be important for their catalytic activities (Gotoh et al., J. Biol. Chem., 273, 17477-17482, 1998; Leung et al., J. Biol. Chem. 273:32408-32415, 1998). This dimerization may facilitate inter-molecular autophosphorylation leading to activation, as is the case for receptor-tyrosine kinases. In some signaling pathways, MAPKKK kinases (MAPKKKKs) function upstream of MAPKKKs. For example, the Ste20 MAPKKKK in budding yeast functions to activate the Ste11 MAPKKK in the mating pheromone pathway (Herskowitz, Cell 80:187-197, 1995). Similarly, Ste20-like kinases are implicated in the activation of MAPKKKs in mammalian cells. Raf-1 is phosphorylated and activated by p21 (Rac/Cdc42)-activated kinase (PAK) (King et al., Nature 396:180-183, 1998). Germinal center kinase (GCK) functions upstream of MEKK1 in the TNF-α signaling pathway leading to JNK activation. However, the molecular mechanism by which IL-1 activates TAK1 is yet to be elucidated.